title: [Novel NADPH-dependent L-aspartate dehydrogenases from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum].
abstract: The genes encoding putative L-aspartate dehydrogenases (EC 1.4.1.21, ADH) from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum were cloned and expressed in Escherichia coli. The respective enzymes in the form of hybrid proteins with N-terminal hexahistidine tags were purified to apparent homogeneity. Both enzymes catalyzed in vitro the reductive amination of oxaloacetate to L-aspartate by an order faster than the reverse reaction at a respective pH optimum of 8.0-9.0 and 9.8; also, the enzymes only catalyzed amination under physiological conditions (pH 7.0-8.0). Their specificity to NADPH was higher by 1-2 orders of magnitude than that to NADH. The apparent KM values of ADHs from R. palustris for oxaloacetate, ammonium, and NADPH at pH 9.0 were 9.2, 11.3, and 0.21 mM, respectively, and the corresponding KM values of ADH from B. japonicum were 21, 4.3, and 0.032 mM, respectively. The amination activity of novel ADHs may be important for the fixation of inorganic nitrogen in vivo and used for the construction of a bacterial strain-producer of L-aspartate by metabolic engineering methods.
authors.forename: T M
authors.initials: TM
authors.lastname: Kuvaeva
authors.forename: Zh I
authors.initials: ZhI
authors.lastname: Katashkina
authors.forename: A D
authors.initials: AD
authors.lastname: Kivero
authors.forename: S V
authors.initials: SV
authors.lastname: Smirnov
mesh_terms: D000586:Amination; D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D020369:Bradyrhizobium / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D009586:Nitrogen Fixation / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012241:Rhodopseudomonas / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010071:Oxaloacetates; D011993:Recombinant Fusion Proteins; D009243:NAD; D001224:Aspartic Acid; D006639:Histidine; D009249:NADP; D000594:Amino Acid Oxidoreductases; C025745:aspartate dehydrogenase